It is difficult to make vaccines against influenza A viruses (IAVs) due to the accumulation of mutations in antibody binding sites on the external proteins of these viruses. The mechanisms that promote this process, termed antigenic drift, are poorly understood. There are fairly effective vaccines for IAVs, but vaccine failure is common due to antigenic drift, and seasonal IAVs contribute to over 30,000 deaths in the US annually. The work outlined in this proposal will elucidate mechanisms involved during antigenic drift of IAVs. The current paradigm of how IAVs undergo antigenic drift is largely based on in vitro and in ovo studies showing that the virus can accumulate sequential mutations when grown in the presence of single monoclonal antibodies. Using a mouse model, we have found that antigenic drift of a classical H1N1 IAV occurs in vivo as the virus adjusts receptor binding avidity when confronted with different levels of sub-neutralizing antibodies. From these studies we propose that antigenic drift primarily occurs as a byproduct of receptor binding modulation rather than directly from mutations that abolish antibody binding per se. In this proposal we will first determine if additional subtypes of IAVs utilize this receptor-modulating mechanism during antigenic drift and we will examine if viral fitness is compromised during this process. To do this, we will use mice to model in vivo antigenic drift of 2 IAVs that circulate in humans (H3N2 and swine-origin H1N1 IAVs). Next, we will continue to use the mouse model to determine if the quality and specificity of anti-IAV antibody responses influence mutation selection in vivo. In these experiments, we will precisely define anti-IAV antibody repertoires that are induced in individual mice following different pre-exposure regiments and we will complete passive transfer studies to determine how quantitatively and qualitatively different antibody responses alter mutant selection. Finally, we will test the hypothesis that receptor diversity promotes antigenic diversity of IAVs. For these studies, we will select variants in vitro in cell lines that have different receptor compositions and we will complete in vivo studies in mice that lack specific IAV receptors. Collectively these studies will increase our understanding of antigenic drift of IAV viruses and will ultimately aid in the rational design of vaccines. The overall career goal of the candidate is to establish an independent academic laboratory that focuses on basic mechanisms of pathogen-host interactions. The K22 award will help the candidate achieve this goal and will further develop the candidate's scientific and professional skills. Project Narrative: Most viral vaccines work by inducing antibodies, which are molecules that prevent pathogens from entering and replicating in cells. Vaccines can be ineffective when viruses acquire mutations that prevent the binding of antibodies. The purpose of this work is to understand how influenza A viruses accumulate these types of mutations.